Cell fate determination during development often requires morphogen transport from producing to distant responding cells. Hedgehog (Hh) morphogens present a challenge to this concept, as all Hhs are synthesized as terminally lipidated molecules that form insoluble clusters at the surface of producing cells. While several proposed Hh transport modes tie directly into these unusual properties, the crucial step of Hh relay from producing cells to receptors on remote responding cells remains unresolved. Using wing development in Drosophila melanogaster as a model, we show that Hh relay and direct patterning of the 3-4 intervein region strictly depend on proteolytic removal of lipidated N-terminal membrane anchors. Site-directed modification of the N-terminal Hh processing site selectively eliminated the entire 3-4 intervein region, and additional targeted removal of N-palmitate restored its formation. Hence, palmitoylated membrane anchors restrict morphogen spread until site-specific processing switches membrane-bound Hh into bioactive forms with specific patterning functions.
General_information_readme
general description of project and description of manuscript files
Schuermann_Figure_1
Schuermann_Figure_1 - contains Prism-File "angles between nearest neighbours" to quantify patterns of immunoggold-labelling of cell-surface associated large Hedgehog (Hh) morphogen clusters. Contains Folder "IEM of Shh on Bosc23" showing original IEM data. Contains PyMol file "apbs_olig_hh.pse" showing a modelled fly Hh pentamer using crystal lattice interactions observed in the human Sonic Hh crystal structure pdb3m1n as a template.
Schuermann_Figure_2
Schuermann_Figure_2 - contains Folders showing Drosophila wing patterning phenotypes as a consequence of Hh or dominant-negative HhC85S over expression under the control of engrailed-Gal4 or patched-Gal4 (described below). Contains Prizm files to quantify the intervein regions between longitudinal wing veins L3-L4 and L2-L3. The obtained ratio serves as a readout for Hh patterning activity. CD8-GFP serves as a control (wild-type patterning).
Schuermann_Figure_3
Schuermann_Figure_3 - contains Excel-data on HhC85S multimerization and demonstrates that HhNC85S, a non-lipidated artificial Hh variant, does not act in dominant-negative manner. Contains Prize-Files to quantify unaffected wing patterning in three independently derived HhNC85S lines. The original wing patterns are shown in folders en>HhNC85S and ptc>HhNC85S.
Schuermann_Figure_4
Schuermann_Figure_4 - contains files demonstrating that the consecutive N-terminal truncation of HhC85S reverses the dominant negative phenotype. Supplemental folders contain confirming data obtained from independent driver lines en(2)-Gal4 and Hh_Gal4. Unimpaired multimerization as a prerequisite for direct contact with, and dominant-negative suppression of Hh activity are shown as excel-files. HhC85SD86-100 denotes a non-palmitoylated Hh variant that lacks inhibitory N-terminal peptide amino acids 86-100.
Schuermann_Figure_6
Schuermann_Figure_6 - contains excel-file showing gel filtration (size exclusion chromatography, or SEC) results. HA-tagged Hh variants are unimpaired in their ability to multimerize.
Schuermann_Figure_7
Schuermann_Figure_7 - shows severe dominant phenotypes (including lethality, see excel-table) of N-terminally HA-tagged Hh variants. Two different en-drivers were used (en> and en(2)) and two independent HAHh lines that integrated at different chromosomal locations. ptc> expression results are also shown, demonstrating severe dominant activities only if expressed in the posterior (Hh-producing) but not anterior (pic-expressing, Hh receiving) compartment. Experiments conducted at different temperatures are shown, because UAS-transgenes are expressed in temperature-dependent manner.
Schuermann_Figure_9
Schuermann_Figure_9 - contains data obtained from the en> or 34B> controlled co-expression of Hh from chromosome two and HA-tagged transgenes, HhC85S and HAHhC85S from chromosome 3. HAHh also suppresses co-expressed Hh activity in these settings, confirming dominant-negative HAHh function in vivo.
